Schizophrenia is a developmental disorder that is hypothesized to include alterations in glutamatergic function, aberrant N-methyl-D-aspartic acid receptor (NMDAR) signaling and altered excitatory/inhibitory (E/I) balance in the medial prefrontal cortex (mPFC). A critical barrier to progress in improving strategies for the treatment and prevention of schizophrenia is a lack of understanding of the role of presynaptic glutamate release onto GABAergic interneurons by intrinsic cortical vesicular glutamate transporter-2 (VGLUT2) synapses in regulating E/I balance and how this may impact the maturation of PV- containing interneurons. Our goal is to improve strategies for the treatment and prevention of schizophrenia by developing and providing an understanding of the role and influence of VGLUT2-mediated glutamate release on the dysfunction of the PV-inhibitory system. Our central hypothesis is that VGLUT2 provides glutamate to neocortical PV+ inhibitory interneurons in the mPFC that is important for the maturation of excitatory/inhibitory (E/I) balance and to working memory. Our objectives are to (1) establish that functional VGLUT2 synapses target the PV+ interneuronal system in mPFC in vivo and in vitro, (2) demonstrate that the loss of VGLUT2 synapses decreases glutamate release at E-I synapses and suppresses inhibitory transmission, and (3) show that conditional VGLUT2 knockout (KO) mice display deficits in working memory. Our expected outcomes will be (1) an understanding that intrinsic cortical VGLUT2 synapses are relevant to E/I balance because they target recurrent inhibitory feedback neurons and exhibit activity-dependent release of glutamate, (2) the knowledge that loss of intrinsic cortical VGLUT2 expression in conditional VGLUT2 KO mice leads to a reduction in GAD67 in PV+ interneurons and suppresses inhibitory synaptic transmission onto pyramidal neurons, and (3) to affirm that VGLUT2-encoded transmission in corticolimbic circuits is critical to working memory. The impact of these results on strategies for treating and preventing schizophrenia that our outcomes will provide is an important step towards therapeutic insight into the cognitive impairment associated with schizophrenia and an understanding of the role and influence of VGLUT2-mediated release on the maturation of the PV-interneuronal system and E/I balance. Aim 1 will test the hypothesis that intrinsic cortical VGLUT2 synapses are relevant to E/I balance because a) they target PV+ GABAergic interneurons, b) they exhibit activity-dependent release of glutamate, and c) their loss suppresses inhibitory synaptic transmission in the mPFC. We will utilize GAD67gfp+ transgenic mice that selectively express EGFP in PV+ interneurons for immunofluorescence and electrophysiologic studies. Aim 2 will test the hypothesis that VGLUT2-encoded excitatory transmission in corticolimbic circuits during development is critical to working memory in adults, by assessing working memory function using the continuous delayed alternation task in a T-maze in two lines of mice where VGLUT2 inactivation occurs early in development and in adolescent mice.